Ureaplasma parvum colonizes human mucosal surfaces, primarily in the respiratory and urogenital tracts, causing a wide spectrum of diseases, from non-gonococcal urethritis to pneumonitis in immunocompromised hosts. Although the basis for these diverse clinical outcomes is not yet understood, more severe disease may be associated with strains harboring a certain set of strain-specific genes. To investigate this, whole genome DNA macroarrays were constructed and used to assess genomic diversity in 10 U. parvum clinical strains. We found that 7.6% of U. parvum genes were dispersed into one or more strains, thus defining a minimal functional core of 538 U. parvum genes. Most of the strain-specific genes (79%) were of unknown function and were unique to U. parvum. Four hypervariable plasticity regions were identified in the genome containing 93% of the variability in the gene pool (UU32-UU33, UU145-UU170, UU440-UU447 and UU527-UU529). We hypothesized that one of them (UU145-UU170) was a pathogenicity island in U. parvum and we characterized it. Thus, we propose that the clinical outcome of U. parvum infection is probably associated with this newly identified pathogenicity island.
An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity.
This study aimed to determine the occurrence of Vibrio parahaemolyticus in cockles (Anadara granosa) at a harvesting area and to detect the presence of virulent strains carrying the thermostable direct hemolysin (tdh) and TDH-related hemolysin genes (trh) using PCR. Of 100 samples, 62 were positive for the presence of V. parahaemolyticus with an MPN (most probable number) value greater than 3.0 (>1100 MPN per g). The PCR analysis revealed 2 samples to be positive for the tdh gene and 11 to be positive for the trh gene. Hence, these results demonstrate the presence of pathogenic V. parahaemolyticus in cockles harvested in the study area and reveal the potential risk of illness associated with their consumption.
Efforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level.
Mycobacteria have been reported to cause a wide range of human diseases. We present the first whole-genome study of a Non-Tuberculous Mycobacterium, Mycobacterium sp. UM_CSW (referred to hereafter as UM_CSW), isolated from a patient diagnosed with bronchiectasis. Our data suggest that this clinical isolate is likely a novel mycobacterial species, supported by clear evidence from molecular phylogenetic, comparative genomic, ANI and AAI analyses. UM_CSW is closely related to the Mycobacterium avium complex. While it has characteristic features of an environmental bacterium, it also shows a high pathogenic potential with the presence of a wide variety of putative genes related to bacterial virulence and shares very similar pathogenomic profiles with the known pathogenic mycobacterial species. Thus, we conclude that this possible novel Mycobacterium species should be tightly monitored for its possible causative role in human infections.
Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.
We investigated the epidemiology and clonality of 175 nonrepetitive methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical specimens collected between 2011 and 2012 in Kinta Valley in Malaysia. Molecular tools such as polymerase chain reaction, pulsed-field gel electrophoresis, and staphylococcal protein A (spa) typing were used. Our study revealed the predominance of three closely related ermA(+) SCCmec type III pulsotypes belonging to spa type t037 (Brazilian-Hungarian clone), which were deficient in the locus F, but positive for the ccrC gene in majority (65.7%) of the MRSA infections in this region. The first evidence of SCCmec type II MRSA in the country, belonging to spa type t2460, was also noted. Although the carriage of pvl gene was uncommon (8.6%) and mostly confined to either SCCmec type IV or SCCmec type V isolates, most of these isolates belonged to spa types t345 or t657, which are associated with the Bengal-Bay CA-MRSA clone. Interestingly, spa t304 and t690 SCCmec type IV pvl(+) were also detected among the MRSA isolates. Data from this study show the rise of uncommon clones among MRSA isolates in Malaysia.
Antiseptics are commonly used for the management of MDR (multiple drug resistance) pathogens in hospitals. They play crucial roles in the infection control practices. Antiseptics are often used for skin antisepsis, gauze dressing, preparation of anatomical sites for surgical procedure, hand sterilization before in contact with an infected person, before an invasive procedure and as surgical scrub.
We report the first case of an immunocompromised adult patient presenting with cervicofacial lymphadenitis due to Mycobacterium haemophilum, confirmed using hsp65 gene sequencing and line-probe assays. In resource-limited settings, especially in developing countries, appropriate culture methods and rapid molecular diagnostic tools such as hsp65 gene sequencing for identification of this organism may not be readily available. This may cause M. haemophilum infections to go unrecognised or lead to delays in diagnosis. Lack of heightened awareness about the potential for this mycobacterial species to cause infections may also contribute to possible underestimation of M. haemophilum cases in the developing world.
The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.
The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
Enterococci are commonly found in humans, animals and environments. Their highly adaptive mechanisms are related to several virulent determinants and their ability to resist antibiotics. Data on the relationship between the esp gene, biofilm formation and antibiotic susceptibility profiles may differ between countries. This cross-sectional study was conducted to determine the proportion of esp gene and biofilm formation among Enterococcus faecalis and Enterococcus faecium clinical isolates. We also investigated the possible association between the esp gene with antibiotic susceptibility patterns and biofilm formation. The isolates were collected from clinical samples and identified using biochemical tests and 16SRNA. Antibiotic susceptibility patterns and a biofilm assay were conducted according to the established guidelines. Molecular detection by PCR was used to identify the esp gene using established primers. In total, 52 and 28 of E. faecalis and E. faecium were identified, respectively. E. faecium exhibited higher resistance rates compared to E. faecalis as follows: piperacillin/tazobactam (100% versus 1.9%), ampicillin (92.8% versus 1.9%), high-level gentamicin resistance (HLGR) (89.3% versus 25.0%) and penicillin (82.1% versus 7.7%). E. faecium produced more biofilms than E. faecalis (59.3% versus 49.0%). E. faecium acquired the esp gene more frequently than E. faecalis (78.6% versus 46.2%). Interestingly, the associations between ampicillin and tazobactam/piperacillin resistance with the esp gene were statistically significant (X2 = 4.581, p = 0.027; and X2 = 6.276, p = 0.012, respectively). Our results demonstrate that E. faecium exhibits high rates of antimicrobial resistance, esp gene acquisition and biofilm formation. These peculiar traits of E. faecium may have implications for the management of enterococcal infections in hospitals. Thus, concerted efforts by all parties in establishing appropriate treatment and effective control measures are warranted in future.
P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74-92 wt% and 4HB monomer composition of 92-99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).
A pair of primers targeting the hlyA gene for Vibrio cholerae which could distinguish the classical from El Tor biotypes was designed and combined with other specific primers for ompW, rfb complex, and virulence genes such as ctxA, toxR, and tcpI in a multiplex PCR (m-PCR) assay. This m-PCR correctly identified 39 V. cholerae from clinical, water and seafood samples. The efficiency of this multiplex PCR (m-PCR) was compared with conventional biochemical and serogrouping methods. One O139 and 25 O1 V. cholerae strains including 10 environmental strains harbored all virulence-associated genes except 1 clinical strain which only had toxR and hlyA genes. Thirteen environmental strains were classified as non-O1/non-O139 and had the toxR and hlyA genes only. The detection limit of m-PCR was 7 x 10(4) cfu/ml. The m-PCR test was reliable and rapid and reduced the identification time to 4 h.
In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.
In this study, vancomycin-intermediate Staphylococcus aureus (VISA) cells carrying vraS and (or) graR mutations were shown to be more resistant to oxidative stress. Caenorhabditis elegans infected with these strains in turn demonstrated lower survival. Altered regulation in oxidative stress response and virulence appear to be physiological adaptations associated with the VISA phenotype in the Mu50 lineage.
Paenibacillus durus strain ATCC 35681T is a Gram-positive diazotroph that displayed capability of fixing nitrogen even in the presence of nitrate or ammonium. However, the nitrogen fixation activity was detected only at day 1 of growth when cultured in liquid nitrogen-enriched medium. The transcripts of all the nifH homologues were present throughout the 9-day study. When grown in nitrogen-depleted medium, nitrogenase activities occurred from day 1 until day 6 and the nifH transcripts were also present during the course of the study albeit at different levels. In both studies, the absence of nitrogen fixation activity regardless of the presence of the nifH transcripts raised the possibility of a post-transcriptional or post-translational regulation of the system. A putative SigA box sequence was found upstream of the transcription start site of nifB1, the first gene in the major nitrogen fixation cluster. The upstream region of nifB2 showed a promoter recognizable by SigE, a sigma factor normally involved in sporulation.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterium codenamed pi9 was selected for the cloning of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway. Here, the cloning of an 1,113 bp fragment of GAPDH gene which covers the 1,002 bp open reading frame by using multiple PCR steps is described. The partial sequence of this gene was PCR amplified by using degenerate primers followed by the cloning of the flanking sequences by inverse and splinkerette PCR techniques. The success in cloning the GAPDH gene by PCR has bypassed the more time consuming genomic library construction and screening method. The full length GAPDH protein was subsequently expressed in E. coli, purified as His-tag protein and confirmed to be catalytically active. This work demonstrated the use of multiple PCR techniques to clone a gene based solely on sequence comparison. It also laid the foundation for further biochemical and structural characterizations of GAPDH from a psychrophilic bacterium by providing a highly purified recombinant protein sample.